Gel spot picker

ABSTRACT

The present invention relates to a gel sheet ( 1 ) for use in a spot picker device wherein said gel sheet is provide with reference marks ( 5 ′- 5 ′″) which can be used to determine if the gel sheet ( 1 ) has been deformed between the stages of scanning the gel sheet and picking spots out of the gel sheet ( 1 ). The present invention also relates to a method of picking spots using such a gel sheet ( 1 ) and reference marks for use with the method.

FIELD OF THE INVENTION

[0001] The present invention relates to sheets of gels and methods forpicking up gel plugs from sheets of gel.

PRIOR ART

[0002] Many biochemical analytical methods, such as 2 dimensionalelectrophoresis, produce results in the form of sheets of gel upon whichthe analysed substances are grouped in spots. Further analyse of thesubstances can be made by cutting out and picking up the cylindricalplugs of gel containing the spots of substances of interest andtransferring the plugs to other devices such as microtitre plates ortest tubes for further analysis.

[0003] In an automated system for picking spots of interest the gel canbe treated with a coloured dye which can be absorbed by the substancesof interest in order to make then visible. The sheet of gel can then bescanned and the results of the scanning analysed by a computer program,which calculates the co-ordinates of the spots. The gel can then bemoved to a spot picking device called a spot picker and the co-ordinatesof the spots of interest can be loaded into the computer which controlsthe spot picker. The computer controls the movement of the spot-picker'sspot-picking bead and directs it to move to the co-ordinates of eachspot that is to be picked up. The plugs of gel containing the spots areextracted from the sheet of gel by lowering a cylindrical hollow needleon the spot-picker head through the liquid covering the gel and the geluntil it reaches the supporting base plate, applying a suction to theupper end of the needle in order to lift the plug off the base plate andinto the needle where it is caught by a pieced bulkhead near the tip ofthe needle. The plug can then be transported by the spot picker head toa position above a well in a micro-titre plate or a test tube and thenejected out of the needle by pressurised liquid applied to the upperpart of the needle. The spot can then be destained. In order to be surethat the correct material is extracted from the sheet of gel the spotpicker head should be positioned to an accuracy of 0.1 mm or better.

[0004] A problem with this method is that it requires complex andexpensive mechanical high precision guides for both the scanner and spotpicker. Furthermore the geometry of the gel can change during transportbetween the scanner and the spot picker. This means that the spots moveto new co-ordinates and leads to incorrect plugs of gel being picked.Additionally the individual destaining of the spots after they have beenlifted out of the gel is time-consuming. The present invention aims toovercome these problems.

SUMMARY OF THE INVENTION

[0005] The present invention solves the problems of prior art devices bymeans of a gel sheet having the features in the characterising part ofclaim 1. A method in accordance with the present invention has thefeatures presented in the characterising part of claim 6. Referencemarks for use with the gel and method have the features presented in thecharacterising part of claim 7. A system for performing the method hasthe features mentioned in claim 10.

[0006] By means of the present invention, simplified guides for thescanner and spot picker may be used, or the use of guides and mountingfixtures can be eliminated, thus reducing the cost of the equipment andspeeding up handling of gels. Additionally, spots can be extracted evenif the geometry of the gel changes between being scanned and spotpicking. Furthermore in embodiments of devices in accordance with thepresent invention it is possible to extract spots even after they havebeen destained.

BRIEF DESCRIPTION OF THE FIGURES

[0007]FIG. 1 shows schematically a view from above of an embodiment of asheet of gel in accordance with the present invention;

[0008]FIG. 2 shows the sheet of gel of FIG. 1 after it has beendeformed;

[0009]FIG. 3 shows an embodiment of a gel scanner in accordance with thepresent invention; and

[0010]FIG. 4 shows an embodiment of a gel picker in accordance with thepresent invention.

DETAILED DESCRIPTION OF EMBODIMENTS ILLUSTRATING THE INVENTION

[0011]FIG. 1 shows a view from above of a sheet of gel 1 in accordancewith the present invention. Gel sheet 1 can, for example, be a sheet ofgel in a frame 2 which has been to used in 2D electrophoresis and whichhas been subsequently stained in order to make the electrophoresedconstituents of samples visible to a scanner, or a sheet of gelcontaining proteins that have been labelled with a dye prior toelectrophoresis. These constituents of samples appear as spots 3 and inorder to analysis them it is necessary to remove the plugs of gelcontaining the spots 3 from the sheet of gel. This is done by scanningthe gel sheet 1 on the bed 7 of a scanner 9 as shown in FIG. 3 andrecording in a storage means such as a computer memory in a controldevice 10 the positions of the spots 3 identified by the scanner 9.These stored positions can then be used later by the control device of aspot picker device. In the present invention, before the scanning isperformed, the sheet of gel 1 is provided with reference marks 5′-5′″which are printed or painted or stuck or applied in any other suitableway onto the surface of the gel or into the gel or onto a surface underthe gel, so that they are visible and detectable by the scanner. Thesereference marks 5′-5′″, follow any deformation of the sheet of gel. Whenthe gel is scanned the imaging software associated with control device10 of the scanner 9 relates the co-ordinates of the spots to thereference marks and the distances (d1, d2, d3) between the referencemarks is also measured. In other words, the position of each spot can becorrelated to the position of the reference marks 5′-5′″. For example,the imaging software can consider that an imaginary line joiningreference mark 5′ to reference mark 5″, represents a base line in theY-axis (shown by a dotted line) and a line joining reference mark 5″ toreference mark 5′″ represents a base line in the X-axis (shown by adashed line). Thus the position of each spot 3 can be related to eachbase line and stored in memory. If desired the entire gel can now bedestained. The gel and the information relating to the positions of thespots stored in the control device 10 can then be transferred to a spotpicker device 11 (see FIG. 4) that is provided with a camera 13. Thecamera 13 is used with imaging software in the spot picker controldevice 10′ to identify the reference marks 5′-5′″ and to measure thedistances (d1′, d2′, d3′) between them. If the distances (d1′, d2′, d3′)between them are the same as those distances (d1, d2, d3) measured bythe scanner and stored in its memory then it can be assumed that the gelsheet 1 has not been deformed and a movable spot picker head 15 can beused to pick up the spots at the positions stored in the memory in thespot picker control device 10′. In order to provide an integrated systemcontrol device 10 and control device 10′ preferably are the same device.If however, as shown in FIG. 2, the gel sheet has been deformed thensome or all of the distances (d1′, d2′, d3′) will be different from thestored distances (d1, d2, d3). This means that the spots 3 will nolonger be at the positions stored in the memory. The spot picker deviceis provided with software that can make a correlation between the oldand new positions of the reference markers 5′-5′″ and then calculate anew position for each spot. For example, if the software calculates thatthe gel has been skewed 5% in both the X- and Y-directions then it canrecalculate new positions for each spot 3 that are also skewed 5%. Thesenew positions can then be used to guide the spot picker head to thespots even if the spots have been destained and are no longer visible.Additionally if after being transferred from the scanner 9 to the spotpicker 11 the orientation of frame 2 with respect to the X-axis andY-axis of the spot picker 11 is different to its orientation withrespect to the X-axis and Y-axis of the scanner, it is possible for thespot picker software to identify the reference markers 5′-5′″ in theimages from the camera 13 and to calculate the positions of the spots inrelation to these reference marks 5′-5′″. This means that no time orfixtures are required to align the frames 2 with the axes of the scanneror spot picker as the software can compensate for any misalignment.

[0012] While the invention has been illustrated by an embodiment usingthree reference marks it is of course possible to use more referencemarks in order to more accurately identify how the gel has beendeformed. In order to obtain the best results, at least one of thereference marks must be situated to one side of a straight line joiningtwo of the other reference marks in order to provide a two-dimensionalframe of reference.

[0013] However, if the gel is in the form of a long thin strip then itis possible to use only two longitudinally separated reference marks ifit can be assumed that any deformations in the narrow transverse axis ofthe gel are negligible. Similarly, if the gel is stuck to a backing sothat it is substantially fixed, then only two reference marks are neededto establish a frame of reference.

[0014] Furthermore the reference marks are not limited to the shapeshown in the figures but may be of any suitable shape e.g. squares,crosses, triangles, alphanumeric symbols etc.

[0015] Depending on the type of scanner and camera used, the referencemarks may be coloured or made of fluorescent material which must stillbe visible after the gel has been treated with a destaining process. Thereference marks may be in the form of labels that have been printed orpainted on one side and have an adhesive on the other side to allow themto adhere to the gel or, if the gel is sufficiently transparent, to anunderlying support. The reference may be coated with different dyes orcolours depending on the wavelength(s) of light used to scan the gel andthe scanning method. If visible light is used then the reference markcould comprise one or more pigment of contrasting colour e.g. white orblack. If excitation UV light of a certain wavelength (e.g. 480 nm, 530nm, 620 nm, etc.) is used to cause dyed spots of interest in the gel tofluoresce at another wavelength (e.g. 530 nm, 590 nm, 680 nm, etc.),then the reference mark can comprise a dye or the same dye used to dyethe samples (e.g. Cy2, Cy3, Cy5, etc.) that fluoresces at the samewavelength. It is also conceivable that the reference mark can containmore than one dye or pigment. For example, a reference mark could beprinted or painted with a mixture containing one or more of thefollowing dyes or pigments: Cy2 dye, Cy3 dye, Cy5 dye, Sypro ruby/reddye, Sypro orange/tangerine dye, magenta pigment (e.g. SPL 21N/JST 18from Radiant Colors), chartreuse pigment (e.g. SPL 17N/JST 10 fromRadiant Colors) or the like. The pigments and dyes can be dissolved in avarnish (e.g. UVF00106 from Akzo Nobel) and then painted or printed ontolabels to form reference marks. Some dyes are almost insoluble invarnish and in that case it is necessary to first dissolve them inanother solvent, such as dimethylsupoxide (DMSO), which mixes very wellwith the varnish. Alternatively the dyes and pigments may be dissolvedin different solvents and applied in sequence to form different layerson the labels. The relative proportions of the different dyes andpigments are preferably chosen so that during scanning the referencemark has substantially the same pixel intensity irrespective of which ofthe normal excitation light wavelengths is used. This would simplify theuse of the same labels for any scanning wavelength and permit multiplescans on the same sample using different excitation wavelengths andusing the same camera to record the results.

[0016] The present invention is not limited to the embodiments describedabove but many changes and modifications may be made without departingfrom the scope of the inventive concept as defined in the followingclaims.

1. Gel sheet for use in an analysis device for the analysis of a samplecharacterised in that said gel sheet (1) is provided with at least tworeference marks (5′-5′″) on or in the gel sheet (1), wherein saidreference marks (5′-5′″) are not comprised of the sample analysed. 2.Gel sheet in accordance with claim 1 characterised in that said gelsheet (1) has at least three reference marks (5′-5′″).
 3. Gel sheet inaccordance with claim 2 characterised in that at least one of saidreference (5′″) marks is situated to one side of a straight line joininganother two of said reference marks (5′, 5″).
 4. Gel sheet in accordancewith any of the previous claims characterised in that said referencemarks (5′-5′″) are detectable after said gel sheet (1) has been treatedwith a destaining process.
 5. Method for picking sample spots (3) out ofa gel sheet (1) characterised by the steps of: a) applying at least tworeference marks (5′-5′), not comprised of the sample, onto the surfaceof, or into, said gel sheet (1); b) measuring in a scanning device thedistances (d1-d3) between said reference marks (5′-5′″) and thedistances between each spot (3) and said reference marks (5′-5′″), andstoring said distances in a memory; c) moving said gel sheet to a spotpicker device; d) measuring in said spot picker device the distances(d1′-d3′) between said reference marks (5′-5′″); e) comparing thedistances (d1′-d3′″) and (d1-d3) in order to calculated how much the gelsheet (1) has been deformed; f) calculating a new position for each spot(3) based on the deformation calculated in step e); and, g) positioningthe spot picker head at, and picking spots (3) at, the new position foreach spot calculated in step f).
 6. Method in accordance with claim 5characterised by the step of destaining said gel sheet (1) beforeperforming step g).
 7. Reference mark for use in a system or method inaccordance with any of the previous claims, characterised in that it isa label able to be placed in or on said gel and wherein it is notcomprised of the sample analysed in said system or method and wherein itcomprises at least one layer containing one or more dyes and/or whereinsaid dye or dyes is/are Cy2 dye and/or Cy3 dye and/or Cy5 dye and/orSypro ruby/red dye and/or Sypro orange/tangerine dye and/or the like. 8.Reference mark in accordance with claim 7 characterised in it comprisesa self-adhesive label.
 9. System for picking spots from a gel comprisinga gel scanner (9), a spot picker device (11) with imaging means such asa camera (13) and a spot picker (15) wherein it comprises controldevices (10, 10′) comprising software for correlating the position ofreference marks (5′-5′″), of the type according to claim 7 or 8 on animage of a gel scanned by said scanner (9) and the images of the samereference marks on the image of said gel produced by said imaging means(13).